The foundation of this FIRST Award is the testing of a novel concept termed the "functional selectivity hypothesis." It posits that the interaction of "atypical" drugs with a single G-protein receptor isoform may cause functional effects as extreme as agonist vs. antagonist (depending on the nature of the involved G-proteins and the type of conformational changes induced by drug-receptor interaction). While this hypothesis is proposed to generalize to all G-protein coupled receptor systems, I propose to test and refine this hypothesis by studying D2-like dopamine (DA) receptors. The first data in support of this notion were result showing that novel hexahydrobenzo.a.phenanthridine dopaminergic ligands [i.e., dihydrexidine (DHX) and its analogs] activated post-synaptic, but not pre-synaptic, D2-like receptors. The current application will focus on mechanistic studies that can provide a firm underpinning for the underlying "functional selectivity hypothesis." The working hypothesis is that DHX and its N-n-propyl analog have full agonist actions at those D2-like receptors coupled to adenylate cyclase, whereas as these drugs are antagonists (or low efficacy partial agonists) at D2 receptors linked to potassium channels. Both DHX and N-p-DHX cause robust inhibition of adenylate cyclase in several models (e.g., inhibition of forskolin-stimulated or D1-mediated cCAMP synthesis/efflux in striatum, pituitary lactotrophs, or D2-transfected C-6 and MN9D clonal cells). Surprisingly, however, these same drugs have little or no effect on D2 receptors known (or presumed) to be coupled to potassium (K+) channels (e.g., they do not inhibit dopamine cell firing or dopamine release, and they induce only weak activation of K+ channels in pituitary lactotrophs). The development of drugs (such as the hexahydrobenzo[a]phenathridines) with functional selectivity may lead to dramatically improved pharmacotherapies by providing opportunities for targeting a subset of receptor-linked events, thus avoiding the undesirable side effects due to widespread activation or blockade of receptor functions. Such drugs would provide "pharmacological scalpels" for perturbing selected aspects. To begin to elucidate the mechanisms of functional selectivity, my experiments will focus on the actions of DHX on D2 receptor functions in rat striatum. The first aim is to collect concentration/response data to determine the potencies and efficacies of DHX and analogs at D2 receptors linked to adenylate cyclase or K+ channels. Superfused striatal slices will be used to compare effects on Da and ACh release (reflecting ion channel activation) with effects on cAMP efflux (an index of adenylate cyclase activation). Autoreceptor-mediated actions on adenylate-cyclase linked DA synthesis will be assessed by kinetic analysis of tyrosine hydroxylase. Aim 2 will extend results from in vitro studies to two in vivo paradigms, cerebral microdialysis (to measure DA, ACh and cAMP overflow) and the gamma-butyrolactone (GBL) model (to measure effects on DA synthesis). Experiments in Aim 3 will compare the pattern of G-protein activation by DHX and typical D2 agonists. Agonist-induced binding of [alpha-32P]GTP to G-proteins isoforms in striatal membranes and MN9D cells will be used as a marker of G-protein activation.